The effect of high concentrations of circulating acetoacetate on the uptake and utilization of glucose-U-14C was investigated in brain of fed and 7-day fasted sheep. Uniformly 14C-labeled glucose and nonlabeled acetoacetate were infused via the jugular vein while blood samples were being taken from the carotid artery and dorsal sagittal sinus. Samples were analyzed for CO2 and its specific activity, glucose and its specific activity, ketone bodies, lactic acid, plasma-free fatty acids, and plasma-immunoreactive insulin. During 1 hour of infusion of 5 mM of acetoacetate/kg/hour, there was a significant decrease (P less than 0.05) in blood glucose concentration and a significant (P less than 0.05) decrease in the uptake of glucose by the brain. Significant (P less than 0.001) arteriovenous differences across the brain in ketone body concentration were observed in both fed and fasted sheep. Also, a significant increase in plasma-immunoreactive insulin and lactate and a decrease in plasma-free fatty acids were observed. These results indicate that hyperketonemia induced hypoglycemia and reduced the uptake of glucose. It also increased the uptake of ketone bodies and increased lactate production by the ovine brain.